High-resolution HLA allele and haplotype frequencies in majority and minority populations of Costa Rica and Nicaragua: Differential admixture proportions in neighboring countries.

The HLA system shows the most extensive polymorphism in the human genome. Allelic and haplotypic frequencies of HLA genes vary dramatically across human populations. Due to a complex history of migration, populations in Latin America show a broad variety of admixture proportions, usually varying not only between countries, but also within countries. Knowledge of HLA allele and haplotype frequencies is essential for medical fields such as transplantation, but also serves as a means to assess genetic diversity and ancestry in human populations. Here, we have determined high-resolution HLA-A, -B, -C, and -DRB1 allele and haplotype frequencies in a sample of 713 healthy subjects from three Mestizo populations, one population of African descent, and Amerindians of five different groups from Costa Rica and Nicaragua and compared their profiles to a large set of indigenous populations from Iberia, Sub-Saharan Africa, and the Americas. Our results show a great degree of allelic and haplotypic diversity within and across these populations, with most extended haplotypes being private. Mestizo populations show alleles and haplotypes of putative European, Amerindian, and Sub-Saharan African origin, albeit with differential proportions. Despite some degree of gene flow, Amerindians and Afro-descendants show great similarity to other Amerindian and West African populations, respectively. This is the first comprehensive study reporting high-resolution HLA diversity in Central America, and its results will shed light into the genetic history of this region while also supporting the development of medical programs for organ and stem cell transplantation.

Single molecule real-time DNA sequencing of HLA genes at ultra-high resolution from 126 International HLA and Immunogenetics Workshop cell lines.

The hyperpolymorphic HLA genes play important roles in disease and transplantation and act as genetic markers of migration and evolution. A panel of 107 B-lymphoblastoid cell lines (B-LCLs) was established in 1987 at the 10th International Histocompatibility Workshop as a resource for the immunogenetics community. These B-LCLs are well characterised and represent diverse ethnicities and HLA haplotypes. Here we have applied Pacific Biosciences' Single Molecule Real-Time (SMRT) DNA sequencing to HLA type 126 B-LCL, including the 107 International HLA and Immunogenetics Workshop (IHIW) cells, to ultra-high resolution. Amplicon sequencing of full-length HLA class I genes (HLA-A, -B and -C) and partial length HLA class II genes (HLA-DRB1, -DQB1 and -DPB1) was performed. We typed a total of 931 HLA alleles, 895 (96%) of which were consistent with the typing in the IPD-IMGT/HLA Database (Release 3.27.0, January 20, 2017), with 595 (64%) typed at a higher resolution. Discrepant types, including novel alleles (n = 10) and changes in zygosity (n = 13), as well as previously unreported types (n = 34) were observed. In addition, patterns of linkage disequilibrium were distinguished by four-field resolution typing of HLA-B and HLA-C. By improving and standardising the HLA typing of these B-LCLs, we have ensured their continued usefulness as a resource for the immunogenetics community in the age of next generation DNA sequencing.

Recipient/donor HLA and CMV matching in recipients of T-cell-depleted unrelated donor haematopoietic cell transplants.

Improving haematopoietic cell transplantation outcomes by selection of an HLA-matched unrelated donor is best practice; however, donor selection by secondary characteristics is controversial. We studied 1271 recipients with haematological malignancies who underwent T-cell-depleted allografts and had complete data on HLA-matching status for six loci (HLA-A, -B, -C, -DRB1, -DQB1, -DPB1) and clinical outcome data. Five-year overall survival was 40.6%. HLA mismatching (at HLA-A, -B, -C, -DRB1, -DQB1) relative risk (RR) 1.22, 95% confidence interval (CI) 1.2-1.5, P=0.033 for 1 mismatch and RR 1.46, 95% CI 1.1-1.9, P=0.009 for >1 mismatch) and CMV mismatching (RR 1.37, 95% CI 1.2-1.6, P<0.001) were significantly associated with inferior survival. Donors aged <30 years showed a trend towards better survival. The multivariate model for mortality, combining CMV and HLA-match status, found an RR of 1.36 (95% CI 1.1-1.7, P=0.003) for HLA matched/CMV mismatched, an RR of 1.22 (95% CI 0.99-1.5, P=0.062) for HLA mismatched/CMV matched and an RR of 1.81 (95% CI 1.4-2.3, P=<0.001) for HLA/ CMV mismatched, compared with the HLA/CMV-matched recipients. These data suggest that HLA and CMV matching status should be considered when selecting unrelated donors and that CMV matching may abrogate the effect of an HLA mismatch.

Further diversity of the 5' promoter region of the MHC class I-related chain B gene.

We have now found a total of 15 individual MICB promoter sequences, varying by combination of 18 polymorphic positions within the MICB minimal promoter sequence. Sequence-based typing and cloning characterized the three new 5' promoter sequences as MICB-P13, MICB-P14 and MICB-P15.

The novel HLA-B*44 allele, HLA-B*44:220, identified by Single Molecule Real-Time DNA sequencing in a British Caucasoid male.

The genomic sequence of the novel HLA-B*44:220 allele identified in a British Caucasoid male.

Assessing the toxic effects of DMSO on cord blood to determine exposure time limits and the optimum concentration for cryopreservation.

BACKGROUND AND OBJECTIVES: Advantages of using cord blood (CB) over other sources of haematopoietic progenitor cells, such as bone marrow, include the ability to cryopreserve and bank the samples until requested for a transplant. Cryopreservation requires the addition of a cryoprotectant to prevent the formation of intracellular ice during freezing. Dimethyl sulphoxide (DMSO) is commonly used at a concentration of 10% (v/v); however, there is evidence to suggest this chemical is toxic to cells as well as to patients after infusion. MATERIALS AND METHODS: The toxic effects of DMSO were assessed through cell viability and in vitro functional assays in fresh and post-thaw CB samples before determining the maximum exposure time and optimal concentration for cryopreservation. RESULTS: A dose-dependent toxicity of DMSO was observed in fresh samples with 40% removing all viable and functional haematopoietic progenitor cells (HPC). In fresh and post-thaw analysis, minimal toxic effect was observed when cryopreservation was delayed for up to 1 h after 10% DMSO addition. After thawing, DMSO washout was superior to dilution or unmanipulated when maintained for long periods (advantage observed 1 h after thawing). Finally, the optimum concentration for cryopreserving CB was found to be 7.5 to 10% with detrimental effects observed outside of this range. CONCLUSION: These results support the use of 7.5-10% as the optimal DMSO concentration and the maximum exposure time should be limited to <1 h prior to freezing and 30 min post-thaw.

Novel alleles of the transforming growth factor ß-1 regulatory region and exon 1.

Transforming growth factor ß-1, encoded by the TGFB1 gene, is a cytokine that plays a central role in many physiologic and pathogenic processes with pleiotropic effects. Regulatory activity for this gene has been shown for 3.0 kb between positions -2665 and +423 from its translational start site. At least 17 TGFB1 regulatory region and exon 1 alleles have been defined on the basis of 18 polymorphic sites. Polymorphisms in TGFB1's regulatory region have been associated with differential levels of expression of this cytokine and to genetic risk in cancer and transplantation. In this report, we present 19 novel TGFB1 regulatory region and exon 1 alleles: p018-p036. Amplification of TGFB1's regulatory region was performed with an in-house protocol, and novel alleles were defined by either allele-specific amplification and/or molecular cloning of the amplicons, followed by sequencing in isolation. Three of these novel alleles (p018, p019, and p020) are shown to be formed by novel combinations of the aforementioned known polymorphic positions. Another 16 novel alleles are shown to carry additional known and unknown single-nucleotide polymorphisms. Polymorphism in TGFB1's regulatory region could have an impact on important processes, including embryogenesis, hematopoiesis, carcinogenesis, angiogenesis, fibrosis, immune responses, and transplantation, making its characterization necessary.

Two novel 5' promoter sequences of the MHC class I-related chain A gene.

In this study, we have characterized two novel polymorphism of the 5' promoter sequence of MICA gene, MICA-P13 and MICA-P14, by sequence-based typing and cloning.

Caspase-8 polymorphisms result in reduced Alemtuzumab-induced T-cell apoptosis and worse survival after transplantation.

Allo-SCT using unrelated donors is a curative treatment for patients with hematological disorders. The best donor is one matched for 10/10 HLA alleles, however studies have shown an additional survival benefit when considering other genetic factors. It has been shown that a six-nucleotide insertion/deletion polymorphism in the CASP8 gene promoter results in reduced susceptibility of T lymphocytes to undergo apoptosis. In 186 SCT recipients, we found a significantly better OS in those who received a transplant from a WT/WT donor compared with donors with a deletion (3 years: 52 vs 34%; P=0.03; multivariate analysis; RR 0.61; 95% CI 0.38-0.98, P=0.04). This was more marked when both the patient and the donor had a deletion (3 years OS: 62% compared with 36%, P=0.01). As the majority of these patients received Alemtuzumab during conditioning, we went on to analyze the in vitro effect of the polymorphism on Alemtuzumab-induced apoptosis. We showed statistically significantly higher percentages of apoptotic naïve CD4 (P<0.0005) and CD8 (P<0.0005) T cells in WT/WT donors in comparison with donors with a deletion. These data imply an unrecognized role for the CASP8 promoter polymorphism on survival following unrelated SCT particularly in the context of T-cell depletion with Alemtuzumab.

A novel HLA-A allele, A*74:23, identified in an individual from Costa Rica.

The new HLA-A*74:23 allele differs from the closest allele A*74:01 by a nucleotide change in exon 3 at codon 97.

Diversity and characterization of polymorphic 5' promoter haplotypes of MICA and MICB genes.

The major histocompatibility complex (MHC) class I-related chain A (MICA) and B (MICB) are ligands for the natural killer group 2, member D (NKG2D) activating receptor expressed on natural killer (NK) cells, natural killer T (NKT) cells, CD8+ T cells and γδ T cells. Natural killer group 2, member D (NKG2D) ligand expression is stress-related and upregulated by infected or oncogenic cells leading to cytolysis. MICA and MICB genes display considerable polymorphism among individuals and studies have investigated allelic association with disease and relevance of MICA in transplantation, with variable success. It is now known that promoters of MICA and MICB are polymorphic with some polymorphisms associating with reduced expression. We sequenced International Histocompatibility Workshop (IHW) cell line DNA to determine promoter types and alleles encoded by exons 2-6. We found 8 of 12 known MICA promoter polymorphisms and although promoter P7 dominated, other promoters associated with the same allele. For example, MICA*002:01 had promoters P3, P4 or P7 and the common MICA*008:01/04 type had P1, P6 or P7. Similarly, we sequenced 8 of 12 known MICB promoter haplotypes. Some coding region defined MICB alleles had a single promoter, for example, MICB*002:01 and promoter P9, whereas the promiscuous MICB*005 allele had promoters P1, P2, P5, P6, P10 or P12. The results indicate potential for variation in expression of MICA and MICB ligands between individuals with the same allelic types. If differential expression by polymorphic MICA and MICB promoters is confirmed by functional studies, involvement of these genes in disease susceptibility or adverse transplantation outcomes may require knowledge of both promoter and allelic types to make meaningful conclusions.

Genomic sequence of the rare HLA-A*02:95 allele identified by sequence-based typing and cloning.

Genomic sequence of HLA-A*02:95 identified in an Anthony Nolan volunteer donor.

Further polymorphism of the RAET1E/ULBP4 gene in humans.

Description of a novel RAET1E/ULBP4 allele characterized by sequence-based typing and cloning: RAET1E*011.

A review of the haematopoietic stem cell donation experience: is there room for improvement?

Donation of haematopoietic stem cells, either through BM or PBSC collection, is a generally safe procedure for healthy donors although adverse reactions are a definite risk. The invaluable source of donation and its central role in transplantation implies that every effort should be made to alleviate possible difficulties the donor encounters. The physical and psychological reactions to donation have been established for some time, but less is known about the factors that are associated with a poorer donation experience. In this article, we provide an overview of the physical and psychological donation experience and focus attention on demographic, physical and psychological factors that may influence this donation experience. Understanding that toxicity profiles vary with certain donor characteristics is crucial as this knowledge could influence practice in numerous ways including the modification of joining and recruitment policies and the improvement of supportive measures and donor follow-up procedures. Although this review deals with both unrelated and related donors (RDs), there is a relative paucity of regulation of RD care and we call for more attention to this area. Owing to the relative rarity of donation in each country, a global effort to collect donor outcome data is needed.

Ethnicity, length of time on the register and sex predict donor availability at the confirmatory typing stage.

Despite over 20 million unrelated donors being listed worldwide, donor attrition at the confirmatory typing (CT) stage of donor acquisition is a key source of delay. Anthony Nolan undertook a study of CT requests from 2010 to 2011 to identify factors associated with attrition. Of 7541 CT requests, 38.2% were cancelled for donor reasons. Of these, 19.4% were personal, 34.1% medical, 36% no contact, 7.9% emigrated and 2.6% others. African (odds ratio (OR) 2.78, P<0.001), African-Caribbean (OR 3.07, P<0.001), Asian (OR 2.65, P<0.001), Jewish (OR 1.54, P=0.009) and Mediterranean (OR=2.38, P<0.001) donors were more likely not to be available compared to Caucasian donors. Female donors were also more likely not to be available (OR=1.32, P<0.001): primarily due to pregnancy. Older donors were less likely to be available in univariate analysis, but this association was not significant after controlling for other factors. Blood donors and those recruited within the past five years had lower rates of attrition. Accumulation of additional attrition-associated characteristics for a given donor was associated with progressively greater odds of attrition (OR 1.99, 2.52, 3.4 and 5.53, respectively, for 1, 2, 3 and 4 risk factors, P<0.001). Donor registries must develop evidence-driven strategies to recruit and retain the most reliable donors.

Uptake and use of recommendations for the diagnosis, severity scoring and management of chronic GVHD: an international survey of the EBMT-NCI Chronic GVHD Task Force.

In 2005, the National Institutes of Health (NIH) consensus conference published a series of papers recommending methods to improve the conduct of clinical trials in chronic GVHD. Although the NIH recommendations were primarily aimed at strengthening research, several papers addressed issues relevant for clinical practice, particularly diagnosis, severity scoring, and ancillary and supportive care practices. We conducted an international survey to assess the uptake of these recommendations, identify barriers to greater use and document the use and perceived effectiveness of available treatments. The response rate for the American survey of 1387 practitioners was 21.8%, and it was 24.6% for 407 centers surveyed in Europe, Asia, Australia and Africa. Most respondents were familiar with the NIH consensus recommendations (94-96%) and used them in practice. Multiple barriers to greater use were reported. Besides lack of time (55-62%), unfamiliarity with the recommendations, scarcity of evidence supporting the impact of recommendations on outcomes, insufficient training/experience in chronic GVHD management and inaccessibility of subspecialists were also endorsed. Systemic corticosteroids were reported to be the most effective treatment for chronic GVHD, but many others were perceived to have moderate or great success. Therapeutic management of steroid-refractory chronic GVHD was identified as the highest priority for research.

Instantaneous Position and Orientation of the Body Segments as an Arbitrary Object in 3D Space by Merging Gyroscope and Accelerometer Information / Posición y orientación instantánea de los segmentos corporales como un objeto arbitrario en el espacio 3D a través de la fusión de la información de acelerómetros y giroscopios

This work presents an algorithm to determine instantaneous orientation of an object in 3D space. The orientation was determined by using a Direction Cosine Matrix (DCM), performed by the combination of three consecutive rotations, around each to the main axes of the evaluated system, using quaternions. An inertial measurement unit (IMU), consisting of 3 axes gyroscope and 3 axes accelerometer was used in order to establish 2 coordinate systems; The first one describes object movement, by using gyroscope as a main source of information, relating the angular rate of change along time. The second defines a coordinate reference system, relating the acceleration of gravity to an inertial direction vector. A proportional integral (PI) feedback controller was used, which includes sensors information, eliminating offset, cancelling drift and improving the accuracy of the orientation. The proposed algorithm can be used for assessing both the position and orientation of the body segments which is very important in orthopedic, traumatology and rheumatology important for diagnosis, prognostic, therapeutic, research and as well as the design and fabrication of measuring devices used in surgical instrumentation, prostheses and ortheses. It is important to note that the developed system opens the opportunities to be implemented on ambulatory joint evaluation through a wearable system, due to the dimensions and requirements of the sensors.

El presente trabajo presenta un algoritmo para determinar la orientación instantánea de un objeto en el espacio 3D. La orientación fue determinada utilizando una matriz de cosenos directores (DCM) conformada por la combinación de 3 rotaciones consecutivas alrededor de cada uno de los ejes del sistema evaluado, utilizando cuaterniones. Una unidad inercial de medida (IMU) compuesta por un giroscopio de 3 ejes y un acelerómetro de 3 ejes fue utilizada con el objetivo de establecer 2 sistemas coordenados; Un sistema coordenado describiendo el movimiento del objeto, utilizando al giroscopio como fuente principal de información, estableciendo la relación de cambio con respecto al tiempo. Un sistema coordenado de referencia, relacionando la aceleración gravitacional a un vector inercial. Un control por retroalimentación proporcional integral (PI) fue utilizado con el objetivo de combinar la información de los sensores, eliminando las desviaciones por offset y deriva, mejorando la precisión en la orientación. Dadas sus características, el algoritmo propuesto permite su utilización en la evaluación de la posición y la orientación de los segmentos corporales, siendo de suma importancia en ortopedia, traumatología y reumatología para la determinación de diagnósticos, pronósticos terapéuticos e investigación así como el diseño y fabricación de dispositivos de medición, instrumentación quirúrgica, prótesis y ortesis. Cabe destacar que el sistema desarrollado abre oportunidades de ser implementado en el diseño de sistemas ambulatorios de evaluación de las articulaciones, mediante el uso de elementos transportables dadas las reducidas dimensiones y limitaciones de los sensores empleados.

HLA-DRB1*03:49, a novel allele identified by group-specific sequence-based typing in a North European individual.

Identification of the antigen presenting molecule HLA-DRB1*03:49 by group-specific sequence-based typing.